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. 2006 May 23;103(21):8191-6.
doi: 10.1073/pnas.0602902103. Epub 2006 May 15.

The TodS-TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins

Jesús Lacal  1 , Andreas Busch, María-Eugenia Guazzaroni, Tino Krell, Juan L Ramos
Affiliations

The TodS-TodT two-component regulatory system recognizes a wide range of effectors and works with DNA-bending proteins

Jesús Lacal et al. Proc Natl Acad Sci U S A. .

Abstract

The TodS and TodT proteins form a previously unrecognized and highly specific two-component regulatory system in which the TodS sensor protein contains two input domains, each of which are coupled to a histidine kinase domain. This system regulates the expression of the genes involved in the degradation of toluene, benzene, and ethylbenzene through the toluene dioxygenase pathway. In contrast to the narrow substrate range of this catabolic pathway, the TodS effector profile is broad. TodS has basal autophosphorylation activity in vitro, which is enhanced by the presence of effectors. Toluene binds to TodS with high affinity (Kd = 684 +/- 13 nM) and 1:1 stoichiometry. The analysis of the truncated variants of TodS reveals that toluene binds to the N-terminal input domain (Kd = 2.3 +/- 0.1 microM) but not to the C-terminal half. TodS transphosphorylates TodT, which binds to two highly similar DNA binding sites at base pairs -107 and -85 of the promoter. Integration host factor (IHF) plays a crucial role in the activation process and binds between the upstream TodT boxes and the -10 hexamer region. In an IHF-deficient background, expression from the tod promoter drops 8-fold. In vitro transcription assays confirmed the role determined in vivo for TodS, TodT, and IHF. A functional model is presented in which IHF favors the contact between the TodT activator, bound further upstream, and the alpha-subunit of RNA polymerase bound to the downstream promoter element. Once these contacts are established, the tod operon is efficiently transcribed.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Induction of PtodX by TodS–TodT in response to a wide range of aromatics. P. putida DOT-T1E bearing pMIR77 (PtodX:′lacZ) was grown on M9 medium with 1 mM of the indicated effector. When turbidity of the cultures was 0.8, β-galactosidase activity was determined. Tested compounds that did not induce were as follows: o-xylene, m- and p-ethyltoluene; o-, m-, and p-nitrotoluene; o-chloro-, o-, m-, and p-iodotoluene; propyl-, butyl-, and isobutylbenzene; 1,2,3-, 1,3,5-, 1,2,4-trimethylbenzene; and benzamide. T, toluene; B, benzene.
Fig. 2.
Fig. 2.
Isothermal titration calorimetry data for the binding of toluene to TodS and its recombinant fragments NTodS and CTodS. (Upper) Heat changes are shown. (Lower) Integrated peak areas are shown. (A) Titration of buffer with 1 mM toluene. (B) Titration of 15 μM TodS with 1 mM toluene. (C) Titration of 12 μM NTodS with 0.8 mM toluene. (D) Titration of 8 μM CTodS with 1 mM toluene. Integrated peak areas are shown for heat changes in B (E) and C (F).
Fig. 3.
Fig. 3.
The catalytic properties of TodS. (A Upper) Autophosphorylation in the presence and absence of toluene. Experiments were carried out in parallel, and the graph (A Lower) shows the densitometric analysis of the gels. (B) Dephosphorylation kinetics. (C) Transphosphorylation to TodT. Conditions for these assays are described in Materials and Methods.
Fig. 4.
Fig. 4.
Identification of TodT and of IHF-binding sites at PtodX. DNase I footprints of IHF (A) and TodT (B). Brackets indicate the protected area. (C) Sequence of PtodX. The gray shading indicates the region protected by TodT. The dotted outline boxes indicate the two TodT-binding sites. Arrows represent palindromic sequences; the asterisk marks a hyperreactive G; sequences matching the IHF consensus sequence are in bold. The narrow box indicates the fragment protected by IHF. The transcription start site is indicated by an arrowhead, and the −10 base pair position in the promoter is marked with a dot and underlined. (D) Sequence alignment of the two TodT binding sites. Conserved nucleotides are boxed.
Fig. 5.
Fig. 5.
Specific binding of TodT to PtodX EMSA for the binding of TodT to a 352-bp DNA fragment containing the PtodX promoter. F, free DNA.
Fig. 6.
Fig. 6.
In vitro transcription from PtodX. Transcription assays were performed as described in Materials and Methods. The assay performed in the presence of 1.5 μM TodS and TodT, 50 nM IHF, and 150 μM toluene is shown in lane 1. In lane 4, TodTD57A replaced TodT. IHF and toluene were omitted in lanes 2 and 3, respectively.
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